Firefly luciferase gene: structure and expression in mammalian cells.

Abstract

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both Si nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell Unes stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30-to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.The luciferase isolated from the common North American firefly Photinus pyralis is one of the most extensively studied of the enzymes that catalyze light production in bioluminescent organisms (for reviews, see references 8, 35, and 36). P. pyralis luciferase has an apparent molecular weight of 62,000 (59) and requires luciferin, ATP, and 02 as substrates. The structure of firefly luciferin has been determined, and the chemical synthesis of this heterocyclic carboxylic acid has been reported (4, 57). The reactions catalyzed by firefly luciferase are: Mg2+ luciferase + luciferin + ATP * -luciferase luciferyl-AMP cDNA library (10). The cDNA hybridized to a single firefly lantern poly(A)+ RNA species estimated to be 1.95 kilobases (kb) in length. The largest cDNA clone isolated, XLuc23, contained -1.8 kb of luciferase cDNA consisting of two EcoRI fragments (the terminal EcoRI sites were added as synthetic linkers during the construction of the cDNA library). This cDNA was inserted into an Escherichia coli expression plasmid and expressed a fusion protein in E. coli that exhibited the ATP-and luciferin-dependent lightemitting activity of firefly luciferase. luciferase luciferyl-AMP + 02 -+ luciferase + oxyluciferin + AMP + C02 + hv The first reaction is the formation of an enzyme-bound luciferyl-adenylate. During the second reaction, the luciferyl-adenylate undergoes an oxidative decarboxylation which results in the production of CO2, oxyluciferin, AMP, and light. When excess substrates are added to firefly luciferase the reaction produces a flash of light that is proportional to...

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“…After 48 h cell extracts were prepared in lysis bu er (10 mM HEPES pH 7.9, 400 mM NaCl, 0.1 mM EGTA, 0.5 mM DTT, 5% glycerol, 0.5 mM PMSF) and then chloramphenicol acetyltransferase and luciferase activities were determined (de Wet et al, 1987;Gorman et al, 1982).…”

The human dopamine D4 receptor repeat sequences modulate expression

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Intracellular rate-limiting steps of gene transfer using glycosylated polylysines in cystic fibrosis airway epithelial cells

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